Human milk contains a remarkable content and structural diversity of oligosaccharides (HMO) that act as a component of the innate immunity by preventing attachment of pathogens to the intestinal lining and by promoting colonization by a healthy microbiota. Despite their recognized importance in neonatal health, research on HMO has been hindered by insufficient quantities to conduct feeding studies. Our long range goal is to understand the mechanisms whereby HMO stimulates neonatal innate and adaptive immunity. We are ideally positioned to undertake these investigations as we have access to large quantities of enzymatically synthesized lacto-N-neotetraose (LNnT) and human milk from which to extract HMO. The objective of this application is to determine how HMO, LNnT, polydextrose and galactooligosaccharide (PDX/GOS) influence activation of dendritic cells (DC) and lymphocyte responses to vaccination against rotavirus (RV) and a subsequent RV challenge in the piglet model. Our central hypothesis is that HMO will enhance immune function by two inter-related mechanisms by directly interacting with DCs and, indirectly, by altering the intestinal microbiota. First, we postulate that HMO glycans interact with c-type lectin receptors on DC, which induce in DC activation and activation of B- and T-lymphocytes. Second, we hypothesize that HMO will promote the growth of Lactobacillus and Bifidobacterium, leading to fermentation of oligosaccharides and enhanced mucosal resistance to RV infection. The specific aims will 1) assess the potential for HMO, LNnT and PDX/GOS to be fermented, to stimulate DC activation and to inhibit RV infectivity and binding in vitro, 2) evaluate the prebiotic actions of oligosaccharides on microbial colonization, mucosal immunity and the response RV vaccination in the piglet, and 3) establish how oligosaccharides alone or in combination with vaccination modulate the response to RV infection in the piglet. The rationale for undertaking this research is that no previous studies have systematically investigated how HMO, LNnT and synthetic prebiotics modulate the microbiota and mucosal immunity. The use of relevant preclinical animal model of RV vaccination and challenge that will further our understanding of the mechanisms underlying the disease and demonstrate the potential for HMOs to improve intestinal function, modulate host-microbe interactions and alter mucosal immunity. Reducing the morbidity and mortality from RV will decrease health care costs and improve the quality of life for infants worldwide.